Summary of Work: Mismatch repair contributes to genetic stability by eliminating DNA biosynthetic errors that arise during replication and by controlling the level of genetic recombination. This proposal seeks to define the mechanism(s) by which E. coli MutS and MutL block recombination between closely related but non-identical DNAs. At the present time studies have primarily focused on the role of MutS ATPase activity and MutL on strand transfer. In replication fidelity MutS initates repair by binding the mismatch site. The exact function of MutL is not known. Notwithstanding, we do know MutL is required in an early step of mismatch repair. Current models suggest that MutL adds to the MutS-mismatch complex in an ATP-dependent fashion. MutS has within its c-terminal domain a nucleotide binding concensus sequence responsible for ATP hydrolysis. The exact role of ATP binding/hydrolysis in methyl-directed repair or recombination is not known. We now know that nucleotide binding by MutS results in loss of mismatch recognition. Consequently it is highly probable that ATP hydrolysis is the driving force behind MutS-depedent DNA translocation along the DNA helix contour. To help clarify the biochemistry behind MutS ATPase activity we have carried out site-directed mutagenesis to define the active site residue (carboxylate) responsible for hydrolysis. We have preliminary evidence to suggest aspartic acid 651 is the active site residue. Currenlty, we are characterizing several other residues that also might participate in hydrolysis and testing their ability to bind a G/T mismatch. Previously our lab investigated the dominant negative effect associated with two MutS mutants in vitro. We were, however, unable to dissect the genetics behind this dominant negative phenotype in vivo with our given biochemical strategies. Therefore we decided to tackle this problem utilizing an approach that would allow us to prepare heterodimers between mutant and wild-type MutS. This procedure involved differentially GST- and his6- fusions proteins. The separate tags facilitated rapid and pure 1:1 mutant-wild-type heterodimers. We are presently characterizing this heterodimer for ATPase activity, mismatch recognition and repair. Examination of mismatch repair in strand transfer revealed MutS and MutL proteins along with RuvAB complex act to destroy homeologous recombination intermediates generated by recA in vitro. This effect was mismatch dependent, as branched intermediates between M13-M13 were unimpeded by these activities. This is the first evidence to demonstrate an anti-recombination activity with RuvAB strand transferase.